Regulation of cell growth by methylglyoxal in Dictyostelium discoideum

Abstract

The intracellular methylglyoxal (MG) is regulated by conversion system containing glutathione (GSH) or aldose reductase (ALR) and is an endogenous metabolite inhibiting cell growth. The γ-glutamylcysteine synthetase (GCS) null mutant of Dictyostelium discoideum showed cell growth retardation and G1 arrest following GSH deprivation. To investigate the precise mechanism of the growth arrest induced by GSH depletion, proteomic analysis of gcsA¯ cells during GSH depletion was performed. Aldose reductase 1 (AlrA) was the most prominently induced protein in gcsA¯ cells and its induction was dependent on GSH concentration. During GSH depletion, gcsA¯/alrAas cells exhibited significantly decreased growth and stimulated cell cycle arrest. In gcsA¯ cells, MG level gradually increased as GSH concentration decreased. The gcsA¯/alrAas cells had higher MG level at all different concentrations of GSH and MG level dramatically increased after GSH depletion. These results indicate that depletion of GSH induces MG accumulation and AlrA plays an important role in regulating intracellular MG level. In the media supplemented with different concentrations of MG, KAx3/alrAas cells showed more delayed growth and more increased G1 phase proportion of cells than those of wild-type KAx3. These data support that MG accumulation by GSH depletion is a primary factor inducing cell cycle arrest. The polycationic polyamine as diamine putrescine (PUT), triamine spermidine (SPD) and tetraamine spermine (SPM) are essential factors for growth in all living cells. When PUT and MG reaction were first performed using UV-visible sperctra, new absorption band was produced at 327 nm. Dictyostelium wild-type KAx3 growth was regulated by PUT. To evaluate the in vivo function of PUT in Dictyostelium, ornithine decarboxylase (ODC), converting PUT from ornithine, was disrupted and overexpressed. During PUT depletion, the growth of odc¯ cells decreased and G1 arrest. Then, growth of the odc¯ cells arrested by PUT depletion was restored when 1 mM PUT was replenished again. These result showed that odc¯ cells was PUT auxotroph and PUT regulated cell growth in Dictyostelium. To examine in vivo MG regulation by PUT, PUT level of gcsA¯/alrAas cells significantly decreased despite increased mRNA level odc during GSH depletion. Then, mRNA level of gloA gene encoding glyoxalase I, level of intracellular GSH and MG was increased in odc¯ cells during PUT depletion. When wild-type KAx3 and odcoe cells cultured with different concentration MG, odcoe cells overcame MG toxicity compared with wild-type KAx3. FACS analysis showed that the proportion of G1 phase in odcoe cells were more decreased than that of wild-type KAx3. These results demonstrate that PUT directly regulates intracellular MG. Because GSH and PUT regulated cell growth and intracellular MG, depleted gcsA¯ and odc¯ cells cultured with put together by replacing the products as PUT and GSH, respectively. Interstingly, cell growth and cell cycle recovered in both gcsA¯ cells culutred with GSH and odc¯ cells culutred with PUT. Taken together, these data suggested that MG accumulated by GSH and PUT depletion is a primary factor to regulate cell growth in D. discoideum.